Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na⁺ channel (ENaC). ENaC is composed of three subunits (α, β, γ), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of β- or γ-ENaC. We recently demonstrated that the ubiquitin–protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of β-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na⁺ reabsorption by the distal nephron, and hence the hypertension. J. Clin. Invest. 103:667–673 (1999)

Rationale:: The cardiac sodium channel Na _v 1.5 plays a key role in excitability and conduction. The 3 last residues of Na _v 1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin–dystrophin complex. As dystrophin is absent at the intercalated discs, Na _v 1.5 could potentially interact with other, yet unknown, proteins at this site. Objective:: The aim of this study was to determine whether Na _v 1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results:: Immunostaining experiments demonstrated that Na _v 1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Na _v 1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Na _v 1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Na _v 1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current ( I _Na ) measured by patch-clamp. The I _Na generated by Na _v 1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Na _v 1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. Conclusions:: These data support a model with at least 2 coexisting pools of Na _v 1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97.

The cardiac sodium channel Na _v 1.5 plays a key role in cardiac excitability and conduction. The purpose of this study was to elucidate the role of the PDZ domain-binding motif formed by the last three residues (Ser-Ile-Val) of the Na _v 1.5 C-terminus. Pull-down experiments were performed using Na _v 1.5 C-terminus fusion proteins and human or mouse heart protein extracts, combined with mass spectrometry analysis. These experiments revealed that the C-terminus associates with dystrophin, and that this interaction was mediated by alpha- and beta-syntrophin proteins. Truncation of the PDZ domain-binding motif abolished the interaction. We used dystrophin-deficient mdx ^5cv mice to study the role of this protein complex in Na _v 1.5 function. Western blot experiments revealed a 50% decrease in the Na _v 1.5 protein levels in mdx ^5cv hearts, whereas Na _v 1.5 mRNA levels were unchanged. Patch-clamp experiments showed a 29% decrease of sodium current in isolated mdx ^5cv cardiomyocytes. Finally, ECG measurements of the mdx ^5cv mice exhibited a 19% reduction in the P wave amplitude, and an 18% increase of the QRS complex duration, compared with controls. These results indicate that the dystrophin protein complex is required for the proper expression and function of Na _v 1.5. In the absence of dystrophin, decreased sodium current may explain the alterations in cardiac conduction observed in patients with dystrophinopathies.

Na _v 1.5, the cardiac isoform of the voltage-gated Na ⁺ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na _v 1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na _v 1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na _v 1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na _v 1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2–dependent ubiquitination of Na _v 1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na _v 1.5 and Nedd4-2. Na _v 1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na _v 1.5. Consistent with the functional findings, localization at the cell periphery of Na _v 1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na _v 1.5, suggesting that Nedd4-2 is a specific regulator of Na _v 1.5. These results demonstrate that Na _v 1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na _v 1.5 by decreasing the channel density at the cell surface.